Our broad, long-term objectives are to understand the fundamental properties and operative mechanisms of infectious prion transmission in animals and humans. Specific to this proposal we seek to identify, and understand the role of cellular factors in neurodegeneration. Our Specific Aim is based on the hypothesis, supported by abundant, compelling preliminary data, that in addition to the prion protein (PrP), additional unidentified factors regulate disease susceptibility and pathogenesis. Starting from cloned, persistently infected cells, we isolated clonal derivatives that lost prion infection. The majorityof such cured clones are subsequently re-infectable (susceptible), while a subpopulation is completely resistant to prions, and this resistant phenotype is immutable. Importantly, all resistant clones express PrP. We show this in two independent infectable cell systems. We surmise that this phenotype reflects a genetic change in susceptible compared to resistant cells. We propose to elucidate the identity of these susceptibility factors in multiple susceptible and resistant clones. Our major approach will be to probe transcriptional differences between these two phenotypes. RNAseq will be the standard approach, although we have a significant amount of data from microarray analysis. Gene expression data from clones of each phenotype will be compared to identify differentially expressed genes. We will use gene ontology analysis to assign differentially regulated genes to functional groups and build networks of interacting genes. We will confirm increases or decreases of selected genes using real time PCR. We will assess whether knockdown or over expression of validated differentially regulated genes modifies the phenotypic traits of clones. We will also assess whether expression of susceptibility genes is modified during prion infection of susceptible cells. Despite evidence that misfolded proteins involved in the pathogenesis of a wide variety of neurodegenerative diseases propagate by a prion mechanism, the unequivocal infectivity of prions causing transmissible spongiform encephalopathies set them apart, providing peerless system in which to elucidate general mechanisms and devise integrated therapeutic approaches for a spectrum of related neurodegenerative disorders.